Hi Everybody,
We recently started doing intracellular
staining for cytokines on a FACScan with CellQuest
and are trying to resolve three issues.
1) We see substantial IL-2 and IFN-g positive
cells. Is there a rational, operator-independent
way of setting the markers so that the number of
positives can be identified? Is there software
that will do this?
2) There are a few IL-4 positives and I am not sure that
I want to trust those low percentages? Is there a
better way to detect Th2 cells?
3) Does anybody have experience staining for TNF-beta (or alpha)
or IL-10?
Thanks in advance for any feedback.
Murali Ramanathan
Pharmaceutics
Re these questions:
I know of no software that will do this. I think you just have to set positives on isotype
matched controls. In fact, given that stimulation to induce ic expression of cks ususally causes a
great increase in intracellular non-specific binding, I have found it very necessary to fine-tune
for each experiment.
You just have to trust your Il-4 findings! - provided you have neat profiles for isotype matched
controls and that your unpermabilised cells are not positive relative to their controls.
IL-4 is so transiently produced that even in "Th2" responses you won't see very many. I would
suggest getting a positive control and running that to be sure. I don't know if there is a
BETTER way to detect Th2 cells: the advantage of this system is a single cell profile. If you
have the luxury of large numbers or cloned populations you can of course try ELISAs or PCR:
but again, the potency of IL-4 means that relatively little mRNA is detectable
Dearbhaile O' donnell,
Dept of Medicine and Therapeutics,
University College Dublin